Methods of treating depression and other related diseases

ABSTRACT

The invention relates to methods for treating depression, anxiety, and other related diseases by administering a peptide NMDAR partial agonist.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/507,252, filed Jul. 13, 2011, and U.S. Provisional Application No.61/471,942, filed Apr. 5, 2011, each of which is hereby incorporated byreference in its entirety. This application is also acontinuation-in-part of and claims priority to International ApplicationNo. PCT/US10/51415, filed Oct. 5, 2010, which, claims priority to U.S.Provisional Application No. 61/248,650, filed Oct. 5, 2009, each ofwhich is hereby incorporated by reference in its entirety.

BACKGROUND

The central nervous system (CNS) of mammals employs many nenroactivepeptides to effect specialized signaling within the brain and spinalcord including the neuroactive peptides somatostatin, cholecystokinin,VIP, Substance P, enkephalin, Neuropeptide Y (NPY), Neurotensin, TRB,CCK, and dynorphin, (see generally The Biochemical Basis ofNeuropharmacology, Cooper, Bloom and Roth, 5th ed., Oxford UniversityPress, New York, 1986), The careful elucidation of the complex signalingpathways, which operate in the CNS, has led to identification ofspecific receptors modulated by these neuroactive peptides presentingimportant therapeutic targets for various disorders associated with theCNS.

The N-methyl-D-aspartate (NMDA) receptor (NMDAR), is one such receptorthat has been implicated in neurodegenerative disorders includingstroke-related brain cell death, convulsive disorders, and learning andmemory. NMDAR also plays a central role in modulating normal synaptictransmission, synaptic plasticity, and excitotoxicity in the centralnervous system. The NMDAR is further involved in Long-term potentiation(LTP), LTP is the persistent strengthening of neuronal connections thatunderlie learning and memory (See Bliss and Collingridge, 1993, Nature361:31-39).

Two general classes of glutamate receptors have been characterized inthe central nervous system (CNS). They are the metabotropic glutamatereceptors, which belong to the G-protein coupled receptor family ofsignaling proteins, and the ionotropic glutamate receptors (Muir andLees, Stroke, 1995, 26, 503-513). The ionotropic class is farthersubdivided into the AMPA, kainate, and NMDA receptor subtypes by theselective ligands that activate them.

Ionotropic glutamate receptors contain a ligand-gated ion channel, whichserves as a modulator of synaptic transmission. The NMDA receptor(NMDAR) is unique in that it requires both glutamate and glycine foractivation and subsequent opening of the ion channel (Mothet et al.,Proc. Nat. Acad. Sci., 2000, 97, 4926-4931). Recent studies havedemonstrated that the glycine site can serve to modulate the activity ofglutamate synaptic transmission at the NMDAR. Thus, both the glutamateand glycine sites can be utilized for modulation of NMDAR activity.

The NMDAR is activated by the binding of NMDA, glutamate (Glu), andaspartate (Asp). It is competitively antagonized byD-2-amino-5-phosphonovalerate (D-AP5; D-APV), and non-competitivelyantagonized by phenylcyclidine (PCP), and MK-801. Most interestingly,the NMDAR is co-activated by glycine (Gly) (Kosikowski et al., 1990,Journal of Medicinal Chemistry 33:1561-1571). The binding of glycineoccurs at an allosteric regulatory site on the NMDAR complex, and thisincreases both the duration of channel open time, and the frequency ofthe opening of the NMDAR channel.

Recent human clinical studies have identified NMDAR as a novel target ofhigh interest for treatment of depression. These studies conducted usingknown NMDAR antagonists CPC-101,606 and ketamine have shown significantreductions in the Hamilton Depression Rating Score in patients sufferingwith refractory depression. Although, the efficacy was significant, butthe side effects of using these NDMAR antagonists were severe.

NMDA-modulating small molecule agonist and antagonist compounds havebeen developed for potential therapeutic use. However, many of these areassociated with very narrow therapeutic indices and undesirable sideeffects including hallucinations, ataxia, irrational behavior, andsignificant toxicity, all of which limit their effectiveness and/orsafety.

Further, 50% or more of patients with depression do not experience anadequate therapeutic response to known administered drugs. In mostinstances, 2 or more weeks of drug therapy are need before meaningfulimprovement is observed, as noted in an open-label study onpharmacological treatment of depression. (Rush et al, Am. J. Psychiatry2006, 163: 1905). There currently is no single effective treatment tordepression, anxiety, and other related diseases.

Thus, there remains a need for improved treatments of depression,anxiety and/or other related diseases with compounds that provideincreased efficacy and reduced undesirable side effects.

SUMMARY

The present invention provides methods for treating depression, anxiety,and other related diseases by administering a therapeutically effectivedose of a GLYX peptide or derivative thereof having NMDAR partialagonist activity.

Accordingly, it is an object of the present invention to administercompounds that functionally interact with the glycine site of the NMDARfor the treatment of depression, anxiety, and other related diseases.

In one embodiment, the invention relates to administering adi-pyrrolidine peptide compound comprising the sequence Thr-Pro-Pro-Thr(SEQ ID NO: 13) exemplified by Formula I (GLYX-13) for the treatment ofdepression, anxiety, and other related diseases in mammals includinghumans.

Also provided herein is a method of acutely treating symptoms ofdepression in a patient in need thereof, comprising administering aneffective amount of GLYX-13, for example, in a single unit dose. Suchmethods may relieve the patient of at least one symptom of depressionabout 2 weeks or less, 1 week or less, 1 day or less, 1 hour or less(e.g. 15 minutes or less, half an hour or less, after saidadministration.

Further provided herein is a method of treating refractory depression ina patient resistant to other antidepressants, wherein the patient isadministered an effective amount of GLYX to alleviate at least onesymptom of depression. In certain embodiments, the treatment-resistantpatient is identified as one who has been treated with at least twotypes of antidepressant treatments prior to administration of GLYX-13.In other embodiments, the treatment-resistant patient is one who isidentified as unwilling or unable to tolerate a side effect of at leastone type of antidepressant treatment.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows exemplary GLYX peptides;

FIG. 2 shows the results of Porsolt tests conducted in order to accessantidepressant action of GLYX-13;

FIG. 3 shows the results of Porsolt tests conducted in order to accessantidepressant action of GLYX-13 over a range of concentrations byeither intravenous or subcutaneous administration;

FIG. 4 shows the results of Porsolt tests conducted in order to accessantidepressant action of GLYX-13 administered to the medial prefrontalcortex;

FIG. 5 shows the results of Porsolt tests conducted in order to evaluatethe antidepressant action of GLYX-13 compared to a scrambled peptide;

FIG. 6 shows the results of open field tests conducted in order toaccess anxiolytic action of GLYX-13;

FIG. 7 shows the results of open field tests conducted in order toaccess anxiolytic action of GLYX-13 over a range of concentrations byeither intravenous or subcutaneous administration;

FIG. 8 shows the results of Porsolt tests and Open field tests conductedin order to determine the length of time the antidepressant andanxiolytic action of GLYX-13 persists;

FIG. 9 shows qrt-PCR analysis of the NDMA Cortical mRNA Expressionfollowing Rough-and-Tumble play (RTP);

FIG. 10 shows RTP behavior elicits high rates of 50kHz ultrasonicvocalizations and social defeat elicits high rates of aversive 22kHzultrasonic vocalizations;

FIG. 11 shows effects of GLYX-13 on RTP in rats;

FIG. 12 shows GLYX-13 produced an acute antidepressant-like effect inthe Porsolt test;

FIG. 13 shows the antidepressant-like effects of GLYX-13 were localizedto the medial prefrontal cortex;

FIG. 14 shows GLYX-13 did not exhibit any ketamine-like discriminativestimulus effects or suppress operant responding;

FIG. 15 shows GLYX-13 did not affect prepulse inhibition, or induceconditioned place preference;

FIG. 16 shows GLYX-13 up regulated GluR1 Phosphoserine 845 and itsantidepressant-like effects are blocked by AMPA receptor antagonismsimilar to ketamine;

FIG. 17 shows GLYX-13 acted as a partial agonist at the glycine site ofthe NMDA receptor; and

FIG. 18 shows a plot of cerebrospinal fluid (CSF) concentration ofGLYX-13 versus time after administration.

DETAILED DESCRIPTION

Depression is a common psychological problem and refers to a mentalstate of low mood and aversion to activity. Various symptoms associatedwith depression include persistent anxious or sad feelings, feelings ofhelplessness, hopelessness, pessimism, and/or worthlessness, low energy,restlessness, irritability, fatigue, loss of interest in pleasurableactivities or hobbies, excessive sleeping, overeating, appetite loss,insomnia, thoughts of suicide, and suicide attempts. The presence,severity, frequency, and duration of the above mentioned symptoms varyon a case to case basis. In some embodiments, a patient, may have atleast one, at least, two, at least three, at least four, or at leastfive of these symptoms.

The most common depression conditions include Major Depressive Disorderand Dysthymic Disorder. Other depression conditions develop under uniquecircumstances. Such depression conditions include but are not limited toPsychotic depression, Postpartum depression, Seasonal affective disorder(SAD), mood disorder, depressions caused by chronic medical conditionssuch as cancer or chronic pain, chemotherapy, chronic stress, posttraumatic stress disorders, and Bipolar disorder (or manic depressivedisorder).

Refractory depression occurs in patients suffering from depression whoare resistant to standard pharmacological treatments, includingtricyclic antidepressants, MAOIs, SSRIs, and double and triple uptakeinhibitors and/or anxiolytic drugs, as well non-pharmacologicaltreatments such as psychotherapy, electroconvulsive therapy, vagus nervestimulation and/or transcranial magnetic stimulation. A treatmentresistant-patient may be identified as one who fails to experiencealleviation of one or more symptoms of depression (e.g., persistentanxious or sad feelings, feelings of helplessness, hopelessness,pessimism) despite undergoing one or more standard pharmacological ornon-pharmacological treatment. In certain embodiments, atreatment-resistant patient is one who fails to experience alleviationof one or more symptoms of depression despite undergoing treatment withtwo different antidepressant drugs. In other embodiments, atreatment-resistant patient is one who fails to experience alleviationof one or more symptoms of depression despite undergoing treatment withfour different antidepressant drugs. A treatment-resistant patient mayalso be identified as one who is unwilling or unable to tolerate theside effects of one or more standard pharmacological ornon-pharmacological treatment. In certain embodiments, the inventionrelates to methods for treating refractory depression by administeringan effective amount of GLYX-13 to a treatment-resistant patient in needthereof. In an embodiment, methods of treating depression iscontemplated when a patient has suffered depression for e.g. 5, 6, 7, 8or more weeks, or for a month or more.

GLYX Peptides

As used herein, the term “GLYX peptide” refers to a peptide having NMDARglycine-site partial agonist/antagonist activity, GLYX peptides may beobtained by well-known recombinant or synthetic methods such as thosedescribed in U.S. Pat. Nos. 5,763,393 and 4,086,196 herein incorporatedby reference. Exemplary peptides are illustrated in FIG. 1. In someembodiments, GLYX refers to a tetrapeptide having the amino acidsequence Thr-Pro-Pro-Thr (SEQ ID NO: 13), orL-threonyl-L-prolyl-L-prolyl-L-threonine amide.

For example, GLYX-13 refers to the compound depicted as:

Also contemplated are polymorphs, homology hydrates, solvates, freebases, and/or suitable salt forms of GLYX 13 such as, but not limitedto, the acetate salt. The peptide may be cyclyzed or non-cyclyzed formas further described in U.S. Pat. No. 5,763,393. In some embodiments, ana GLYX-13 analog may include an insertion or deletion of a moiety on oneor more of the Thr or Pro groups such as a deletion of CH₂, OH, or NH₂moiety. In other embodiments, GLYX-13 may be optionally substituted withone or more halogens, C₁-C₃ alkyl (optionally substituted with halogenor amino), hydroxyl, and/or amino. Glycine-site partial agonist of theNMDAR are disclosed in U.S. Pat. No. 5,763,393, U.S. Pat. No. 6,107,271,and Wood et al., NeuroReport, 19, 1059-1061, 2008, the entire contentsof which are herein incorporated by reference.

It may be understood that the peptides disclosed here can include bothnatural and unnatural amino acids, e.g., all natural amino acids (orderivatives thereof), all unnatural amino acids (or derivativesthereof), or a mixture of natural and unnatural amino acids. Forexample, one, two, three or more of the amino acids in GLYX-13 may eachhave, independently, a d- or l-configuration.

Methods

The present invention relates in part to the use of GLYX-13 fortreatment of clinically relevant antidepressant and anxiolytic and fortreatment of depression and anxiety in general.

GLYX-13 may act predominantly at NR2B-containing NMDARs, and may notdisplay the classic side effects of known NMDAR modulators such asCPC-101,606 and ketamine. For example, in vitro studies show thatGLYX-13 can markedly elevate long-term potentiation (LTP) whilesimultaneously reducing long-term depression (LTD) in rat hippocampalorganotypic cultures. In some embodiments, GLYX-13 may produce anantidepressant effect essentially without dissociative side effects whenadministered to a subject in therapeutic amounts. In certainembodiments, an antidepressaqnt effect with essentially no sedation maybe produced by GLYX-13 when administered to a subject in therapeuticamounts. In still other embodiments, GLYX-13 may not have abusepotential (e.g., may not be habit-forming).

In some embodiments, GLYX-13 may increase AMPA GluRI serine-845phosphorylation. In certain embodiments, glycogen, synthase kinase 3β(GSK-3β) may be activated by GLYX-13. In some cases, levels of β-cateninmay be altered after administration of GLYX-13.

Additionally, GLYX-13 may have better Blood-Brain Barrier (BBB)penetration as compared to many of the earlier glycine site ligands(Leeson & Iversen, J. Med. Chem. 37:4053-4067, 1994) and may cross theBBB readily. In some embodiments, GLYX-13 or a composition comprisingGLYX-13 may provide better i.v. in vivo potency and/or brain levelconcentration, relative to plasma levels.

Additionally, GLYX-13 may have a wide therapeutic index compared toother glycine site antagonists such as L-701,324, or other glycine siteantagonists having narrow therapeutic indexes, which result in a verynarrow range of dose between therapeutic effects and ataxia. Forexample, L-701,324 had anticonvulsant effects at doses that producedataxia (Bristow, et al, JPET 279:492-501, 1996). Similarly, a series ofMerz compounds had anticonvulsant effects at doses that produced ataxia.(Parsons, et al., JPET283:1264-1275, 1997).

GLYX-13 may provide a high therapeutic index. For example, GLYX-13 maybe therapeutically effective for depression and/or anxiety with an i.v.or subcutaneous dose range of about 1 to about 10 mg/kg, e.g. about 1mg/kg, about 5 mg/kg, or about 10 mg/kg. In some embodiments, no ataxiaoccurs, at for example a dose of at 500 mg/kg, i.v.

The present invention relates at least in part to the use of a GLYXpeptide or peptides alone or in combination with one or more otherantidepressant treatments, such, as, tricyclic antidepressants, MAO-I's,SSRI's, and double and triple uptake inhibitors and/or anxiolytic drugsfor manufacturing a medicament for treating depression, anxiety, and/orother related diseases including provide relief from depression, anxietyand preventing recurrence of depression, and anxiety. Exemplary drugsthat may be used in combination with a GLYX peptide include Anafranil,Adapin, Aventyl, Elavil, Norpramin, Pamelor, Pertofrane, Sinequan,Surmontil, Tofranil, Vivactil, Parnate, Nardil, Marplan, Celexa,Lexapro, Luvox, Paxil, Prozac, Zoloft, Wellbutrin, Effexor, Remeron,Cymbalta, Desyrel (trazodone), and Ludiomill. It will be appreciatedthat in some embodiments, administration of GLYX-13 may act more quicklythan a co-administered antidepressant treatment, and thus suchco-administration (e.g., administration of GLYX-13 on an acute orimmediate basis, while starting a regimen with another, slower actinganti-depressant at about the same time) may be particularly advantageousin the common situation where the second antidepressant is sloweracting.

Also contemplated herein are methods of treating depression that includeadministering GLYX peptides in combination with (e.g. simultaneonsly orsequentially) other non-pharmacological treatments such aspsychotherapy, electroconvulsive therapy, vagus nerve stimulation and/ortranscranial magnetic stimulation.

A variety of depression conditions are expected to be treated, accordingto this aspect of the invention without affecting behavior or motorcoordination, and without inducing or promoting seizure activity.Exemplary depression conditions that are expected to be treatedaccording to this aspect of the invention include, but are not limitedto, major depressive disorder, dysthymic disorder, psychotic depression,postpartum depression, premenstrual syndrome, premenstrual dysphoricdisorder, seasonal, affective disorder (SAD), anxiety, mood disorder,depressions caused by chronic medical conditions such as cancer orchronic pain, chemotherapy, chronic stress, post traumatic stressdisorders, risk of suicide, and bipolar disorder (or manic depressivedisorder). It should be understood that depression caused by bipolardisorder may be referred to as bipolar depression. In addition, patientssuffering from any form of depression often experience anxiety. Varionssymptoms associated with anxiety include fear, panic, heartpalpitations, shortness of breath, fatigue, nausea, and headaches amongothers. It is expected that the methods of the present condition can beused to treat anxiety or any of the symptoms thereof.

In addition, a variety of other neurological conditions are expected tobe treated according to the methods of the invention. Exemplaryconditions include, but are not limited to, a learning disorder,autistic disorder, attention-deficit hyperactivity disorder, Tourette'ssyndrome, phobia, post-traumatic stress disorder, dementia, AIDSdementia, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, spasticity, myoclonus, muscle spasm, bipolar disorder, asubstance abuse disorder, urinary incontinence, and schizophrenia.

Also provided herein are methods of treating depression in treatmentresistant patients or treating refractory depression, e.g., patientssuffering from a depression disorder that does not, and/or has not,responded to adequate courses of at least one, or at least two, otherantidepressant compounds or therapeutics. For example, provided hereinis a method of treating depression in a treatment resistant patient,comprising a) optionally identifying the patient as treatment resistantand b) administering an effective dose of GLYX-13 to said patient.

Provided herein, in an embodiment, are methods of acutely treatingsymptoms of depression in a patient in need thereof, comprisingadministering an effective amount of GLYX-13, for example, in a singleunit dose. Such methods may relieve the patient of at least one symptomof depression, for about 2 weeks or less, 1 week or less, 1 day or less,or 1 hour or less (e.g. 15 minutes or less, half an hour or less), aftersaid administration. In some embodiments, such methods may relieve thepatient of at least one symptom of depression for about 1 day or more, 1week or more, or 2 weeks or more after said administration. For example,provided herein is a method comprising administering an effective amountof GLYX-13 to a patient suffering from depression, wherein said patientis substantially relieved of at least one symptom of depressionsubstantially earlier after the first administration of GLYX-13, ascompared to the same patient administered a non-GLYX-13 antidepressantcompound. One of skill in the art will appreciate that such methods ofacute administration may be advantageous in a hospital or out-patientsetting.

Symptoms of depression, and relief of same, may be ascertained by aphysician or psychologist, e.g. by a mental state examination. Symptomsinclude thoughts of hopelessness, self harm or suicide and/or an absenceof positive thoughts or plans.

Dosages

The dosage of any compositions of the present invention will varydepending on the symptoms, age and body weight of the patient, thenature and severity of the disorder to be treated or prevented, theroute of administration, and the form of the subject composition. Any ofthe subject formulations may be administered in a single dose or individed doses. Dosages for the compositions of the present invention maybe readily determined by techniques known to those of skill in the artor as taught herein.

A therapeutically effective amount of GLYX peptide required for use intherapy vaires with the form of the depression condition being treated,the length of treatment time desired, the age and the condition of thepatient, and is ultimately determined by the attending physician. Ingeneral, however, doses employed for adult human treatment typically arein the range of about 0.01 mg/kg to about 1000 mg/kg per day. The dosemay be about 0.1 mg/kg to about 100 mg/kg per day. The desired dose maybe conveniently administered in a single dose, or as multiple dosesadministered at appropriate intervals, for example as two, three, fouror more sub-doses per day.

An effective dose or amount, and any possible affects on the timing ofadministration of the formulation, may need to be identified for anyparticular composition of the present invention. This may beaccomplished by routine experiment as described herein, using one ormore groups of animals (preferably at least 5 animals per group), or inhuman trials if appropriate. The effectiveness of any subjectcomposition and method of treatment or prevention may be assessed byadministering the composition and assessing the effect of theadministration by measuring one or more applicable indices, andcomparing the post-treatment values of these indices to the values ofthe same indices prior to treatment.

The precise time of administration and amount of any particular subjectcomposition that will yield the most effective treatment in a givenpatient will depend upon the activity, pharmacokinetics, andbioavailability of a subject composition, physiological condition of thepatient (including age, sex, disease type and stage, general physicalconditions responsiveness to a given dosage and type of medication),route of administration, and the like. The guidelines presented hereinmay be used to optimise the treatment, e.g., determining the optimumtime and/or amount of administration, which will require no more thanroutine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

While the subject is being treated, the health of the patient may bemonitored by measuring one or more of the relevant indices atpredetermined times during the treatment period. Treatment, includingcomposition, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The patient maybe periodically reevaluated to determine the extent of improvement bymeasuring the same parameters. Adjustments to the amount(s) of subjectcomposition, administered and possibly to the time of administration maybe made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The use of the subject compositions may reduce the required dosage forany individual agent contained in the compositions because the onset andduration of effect of the different agents may be complimentary.

Toxicity and therapeutic efficacy of subject compositions may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 and the ED50.

The data obtained from the cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany subject composition lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilised. For compositions ofthe present invention, the therapeutically effective dose may beestimated initially from cell culture assays.

Formulations

The GLYX peptides of the present invention may be administered byvarious means, depending on their intended use, as is well known in theart. For example, if compositions of the present invention are to beadministered orally, they may be formulated as tablets, capsules,granules, powders or syrups. Alternatively, formulations of the presentinvention may be administered parenterally as injections (intravenous,intramuscular or subcutaneous), drop infusion preparations, orsuppositories. For application by the ophthalmic mucous membrane route,compositions of the present invention may be formulated as eyedrops oreye ointments. These formulations may be prepared by conventional means,and if desired, the compositions may be mixed with any conventionaladditive, such as an excipient, a binder, a disintegrating agent, alubricant, a corrigent, a solulblizing agent, a suspension aid, anemulsifying agent or a coating agent.

DNA encoding the GLYX peptides, incorporated into an expression vector,can also be administered, using any of the known administration methods,to express of the GLYX peptides in vivo.

In formulations of the subject invention, wetting agents, emulsifiersand lubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives antioxidants may bepresent in the formulated agents.

Subject compositions may be suitable for oral, topical (including buccaland sublingual), rectal, vaginal, aerosol and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of composition that may be combined with a carriermaterial to produce a single dose vary depending upon the subject beingtreated, and the particular mode of administration.

Methods of preparing these formulations include the step of bringinginto association compositions of the present invention with the carrierand, optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation agents with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrupsor as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), each containing a predetermined amount of a subjectcomposition thereof as an active ingredient. Compositions of the presentinvention may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the subject composition, ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium laurel sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or bydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the subject compositionmoistened with an inert liquid diluent. Tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the subject composition, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubillzing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, cyclodextrins and mixturesthereof.

Suspensions, in addition to the subject composition, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing a subject composition withone or more suitable non-irritating excipients or carriers comprising,for example, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the body cavity and release theactive agent. Formulations which are suitable for vaginal administrationalso include pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate,

Dosage forms for transdermal administration, of a subject compositionincludes powders, sprays, ointments, pastes, creams, lotions, gels,solutions, and patches.

For topical ocular administration compositions of this invention maytake the form of solutions, gels, ointments, suspensions or solidinserts, formulated so that a unit dosage comprises a therapeuticallyeffective amount of the active component or some multiple thereof in thecase of a combination therapy.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise a subject composition in combination with one ormore pharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to uses which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate and cyclodextrins. Proper fluidity may be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

EXAMPLES Antidepressant Action of GLYX-13 in Rats Example 1 Methods

For the evaluation of the antidepressant actions of GLYX-13, the effectof GLYX-13 in the Porsolt test of depression was examined in rats.Porsolt testing is the most commonly used test for assessment ofdepression in animal model, Porsolt testing, also known as Forced SwimTest, is based on a behavioral trait that is sensitive to changes inaffective state. During this test, the animal is placed in a tank (18 cmdiameter×45 cm height) filled to 30 cm with 22-23° C. water. Afterplacing the animal in the tank, the amount of time for which the rat isimmobile when it stops struggling is measured. Porsolt test is based onthe fact that antidepressants increase the latency to immobility anddecrease the time for which the rat is immobile. In most of the cases,this occurs at antidepressant doses that do not increase locomotoractivity on their own. The interpretation of the Porsolt test is thatthe antidepressants reinstate active coping mechanisms and decrease thepassive immobility evoked by stress.

In order to evaluate the effectiveness of GLYX-13 as antidepressant,three month old FBNF1 rats were separated into two groups of 9 animals.The first group of rats was intravenously injected with 10 mg/kg GLYX-13and the second group of animals was intravenously injected with PBSvehicle (1 ml/kg) in a blind manner via chronic subdural femoral veinaccess ports. Both groups of animals were injected 10 to 15 minutesbefore the start of testing.

For Porsolt testing, animals were given a 15 minute pre-test habituationin the tank to induce learned helplessness on day one. Following this,on the subsequent day, the animals were injected with GLYX-13 or vehicle10-15 minutes before being placed into the tank for a 5 minute testsession. After placing the animals in the tank, the amount of time forwhich each animal is immobile when it stops struggling (Mean ImmobilityTime) was measured. Decrease in Mean Immobility Time is a measure of theeffectiveness of the antidepressant.

Results

As shown in. FIG. 2, Mean Immobility Time of the group of animalspretreated with IV injections of PBS Vehicle (1 ml/kg) was highercompared to the Mean Immobility Time of the group of rats pretreatedwith IV injections of GLYX-13 (10 mg/kg) (FIG. 1). GLYX-13 produced anantidepressive-like effect in the Porsolt test with a 56.4±4.6%(Mean±SEM) reduction in floating compared to PBS vehicle (P<0.0001).Therefore, the data from the Porsolt test predicts effectiveness ofGLYX-13 as antidepressant.

Example 2 Methods

A second experiment was performed in the same manner as Example 1,except that 2-3 month Sprague-Dawley rats were used, and treated witheither intravenous (1, 3, 10, 32 mg/kg), subcutaneous (1, 3, 10, 32, 56,100 mg/kg) injections of GLYX-13 or saline vehicle (1 mg/ml) 15 (i,v.)or 20 (s.c.) min before the start of the final 5 min test session.Ketamine (10 mg/kg i.p.) was given 60 min befbre the start of testing(Garcia et al., (2008) Prog Neuropsychopharmacol Biol Psychiatry, 32,140-144). Subcutaneous administration of 10 mg/kg ketamine followed bytesting after 20 min post dosing produced severe dissociative sideeffects, as shown in FIG. 3.

Results

Mean Immobility Time of the group of rats pretreated with IV injectionsor subcutaneous administration of PBS Vehicle was higher compared to theMean Immobility Time of the group of rats pretreated with IV injectionsof GLYX-13 (FIG. 3; 0:127.7±20.4; 2: 84.9±15.0; 3: 66.14±27.53; 1060.6±14.7; 32: 87.19±17.85) GLYX-13 administered intravenously producedan antidepressive-like effect in the Porsolt test at all concentrations,with a 50±10% (Mean±SEM) maximum reduction in floating compared tosaline vehicle (P<0.01). GLYX-13 administered subcutaneously produced anantidepressive-like effect in the Porsolt test at all concentrations,with a 43±7%. (Mean±SEM) maximum reduction in floating compared tosaline vehicle (P<0.01) (0; 142.4±15.6; 1: 135.5±22.0; 3: 100.2±157; 10:76.9±9.7; 32: 84.7.19±11.6) Decrease in Mean Immobility Time is ameasure of the effectiveness of the antidepressant; thus GLYX-13functions as an antidepressant when administered intravenously orsubcutaneously.

Example 3 Methods

To determine whether GLYX-13 acts in an area of the brain that isclinically relevant for depression, GLYX-13 was administered to eitherthe medial prefrontal cortex (MPFC), which is associated with mood anddepression, or to the motor cortex (FIG. 4). Mean time spent immobile inthe Porsolt test was determined for groups of 10 three-month old maleFisher Brown Norway FI (FBNFI) rats pretreated with a single injectionof GLYX-13 (1, 10 ug/side) or a single saline vehicle injection (0.5ul/1 min) bilaterally into the medial prefrontal cortex or motor cortex(dorsal control) in animals with surgically implanted chronic indwellingcannulae aimed at the MPFC. Microinjections were made 1 week aftersurgery, and Porsolt testing was conducted 20 min post injection.Animals received a single 15 min training swim session one day beforedosing.

Results

As shown in FIG. 4, GLYX-13 injected into the MPFC produced anantidepressive-like effect in the Porsolt test with a 50±5% (Mean±SEM)reduction in floating compared to a saline control or to GLYX-13injected into motor cortex (dorsal control). (FIG. 4 0: 115.2±10.89;0.1: 108.77±12.34; 1: 53.46±10.65; 10: 62.54±8.7; Dorsal ControfVehicle: 119.3±10.6; GLYX-13 113.7±13.1; Mean±SEM) These resultsindicate that the antidepressant effect of GLYX-13 may be mediatedthrough the MPFC, an area of the brain associated with mood anddepression.

Example 4 Methods

To determine whether the antidepressant effect of GLYX-13 was specificto the sequence of the GLXY-13 peptide, mean time (sec) spent floatingin the Porsolt test was determined in groups of 12 three-month oldSprague Dawley rats administered with GLYX-13 (TPPT-NH2; 3 mg/kg, i.v.tail vein), a scrambled peptide (PTPT-NH3; 3 mg/kg i.v. tail vein), or0.9% saline vehicle (1 ml/kg, i.v. tail vein). Administration occurred60 min before the 5 min. testing session. Animals received a 15 min.training swim session one day before dosing.

Results

Animals administered GLYX-13 showed a reduction in mean floating time of70±5% (Mean±SEM) compared, fo animals receiving a saline injection, or ascrambled peptide (FIG. 5). Therefore, the antidepressant effect isspecific to GLYX-13 and does not represent an artifact of administeringa peptide generally.

Discussion

The data depicted in FIGS. 2-5 show that GLYX-13 displays significantantidepressant-like properties in rats over a range of doses and routesof administration. In contrast to selective serotonin reuptakeinhibitors (SSRIs), GLYX-13's onset of action was within minutes of asingle dose. These results together with the recent clinical trials ofknown NMDAR molecules, and the fact that GLYX-13 has an outstandingtherapeutic index, especially compared to other modulators of NMDAfunction, make GLYX-13 an attractive candidate for the treatment ofdepression.

These results show that NMDAR glycine-site partial agonists may beexcellent therapeutic candidates for the treatment of depression.

Anxiolytic Action of GLYX-13 Example 5

The purpose of this study was to evaluate action of GLYX-13 on anxiety.In order to gauge the effect of GLYX-13 in rats, the open field test ofanxiety was conducted. The open field test is a widely used neophobictest of anxiety (see, Treit D, Fundytus M. Thigmotaxis as a test foranxiolytic activity in rats. Pharmachol Biochem Behav 1989;31:959-62.).

The open field area used for the open field test generally consists ofan empty and bright square (or rectangular) arena, surrounded by walls.For the current study, an open field of 45 cm×35 cm×20 cm high was used.The rat was placed in the center of the arena and its behavior recordedover a predefined period of time (usually between 2 to 15 min). The openfield test is based on the natural tendency of the rats to avoid openspaces. The open field test is based on the conflict between the innateanxiety that rats have of the central area of the open space versustheir desire to explore new environments. When rats are in an anxiousstate, they have a natural tendency to stay near the walls. This naturaltendency of the rats to stay close to the walls is called thigmotaxis.In this context, anxiety-related behavior is measured by the degree towhich the rat avoids the center of the open field test. Determining thepropensity of the rat to avoid the open field can be determined bymeasuring the number of center crosses made by the rats over apredefined interval of time, or by determining the amount of time therat stays in the center of the field.

In order to evaluate the effectiveness of GLYX-13 as an anxiolytic, twogroups of three month old FBNFI rats were used. The first group wascomposed of 13 rats and the second group was composed of 11 rats. Thefirst group of rats was intravenously injected with 10 mg/kg GLYX-13 andthe second group of rats was intravenously injected with PBS vehicle (1ml/kg) in a blind manner via chronic subdural femoraI vein access ports.Both groups of rats were injected 10 to 15 minutes before the start oftesting. After GLYX-13 injections., both groups of rats were tested inthe open field.

For the open field test, rats were habituated to the open field for 2minutes each day for three consecutive days before the final 2 minutetest session in which GLYX-13 or vehicle was administered. Open fieldactivity was recorded by a video camera mounted above the open fieldarea, and number of center crosses in the open field was measured.

Results

Anxiolytic-like drug effects were measured by increased center crossesin the open field by the rats pretreated with IV injections of GLYX-13(FIG. 6). GLYX-13 (10 mg/kg i.v.) produced an anxiolytic-like effect inthe open field test with a 172.6±34.3% (Mean±SEM) increase in centercrosses compared to vehicle (P<0.005).

Example 6

A second experiment was performed in the same manner as Example 3,except that groups of 8-10 two- to three-month old male Sprague-Dawleyrats pretreated with either intravenous (1, 3, 10, 32 mg/kg),subcutaneous (1, 3, 10, 32, 56, 100 mg/kg) injections of GLYX-13,ketamine (10 mg/kg i.p.) or saline vehicle (1 mg/ml) 15 mm (i.v.), 20(s.c.) or 60 (i.p.) min before the start of the 5 min test session. Eachdata point represents a group of 8-10 rats. The average (mean) amount oftime animals spent in the center of the field was measured.

Results

Anxiolytic-like drug effects were measured by increased center crossesin the open field by the rats pretreated with IV or subcutaneousinjections of GLYX-13 (FIG. 7). Intravenous GLYX-13 administrationproduced an anxiolytic-like effect in the open field test with a 42±5%(Mean±SEM) increase in the amount of time spent in the center of thefield compared to vehicle (P<0.05). Subcutaneous GLYX-13 administrationproduced an anxiolytic-like effect in the open field test with 36±5%(Mean±SEM) increase in the amount of time spent in the center of thefield compared to vehicle (P<0.05).

Discussion

The data reported here show that GLYX-13 displays significantanxiolytic-like properties in rats over a range of doses and routes ofadministration.. The results with GLYX-13 show that NMDAR glycine-sitepartial agonists may be excellent therapeutic candidates for thetreatment of anxiety.

Evaluating Persistence of Action of the Anti-Depressive and AnxiolyticEffects of GLYX-13 Example 7 Methods

To evaluate whether the anti-depressive and anxiolytic effects ofGLYX-13 would persist beyond a few hours, rats were tested in thePorsolt test and the Open Field test, described above. Groups of 10-11three-month old Sprague-Dawley rats were pretreated with GLYX-13 (3mg/kg i.v.), ketamine (10 mg/kg i.v.) or saline vehicle injection (1mg/ml i.v. tail vein) 96 hrs before the start of testing. Animalsreceived a single 15 min. training swim session one day before dosing.Animals were tested 96 hrs post dosing in the Porsolt test without anytesting during the intervening period. Open field testing was conductedwithout pre-habituation 1 day after Porsolt testing (i.e. 120 hrs postdosing). Mean(±SEM) time (sec.) spent floating in the Porsolt test, timespent in the center compartment (sec.), and line crosses in the openfield test were determined.

Results

Results of the Porsolt test showed that animals administered GLYX-13showed an 82%±2 (mean±SEM) reduction in mean floating time compared toanimals administered a saline control (FIG. 8, left panel). Results ofthe open field testing show that animals administered GLYX-13 showed an125%±5 (mean±SEM) increase in mean center time compared to animalsadministered a saline control (FIG. 8, middle panel). Center time is ameasure of anxiolysis. Line crossings are a measure of locomotoractivity, and can be used as a control.

Discussion

These results show that the antidepressant effects of a singleadministration of GLYX-13 last up to 4 days, and that the anxiolyticeffects persist up to 5 days. Thus, while the effects of GLYX-13 can beseen within minutes, the effect of a single administration also persistsfor at least several days.

Understanding the Molecular Underpinning of Rough-And-Tumble Play (RTP)Induced Positive Affect and Evaluation of Hedonic Effect of GLYX-13Example 8 Understanding the Molecular Underpinning of RTP

In order to understand the molecular underpinning of RTP, four RTPsessions of 30 minutes each were conducted with rats. Control rats usedfor this study received identical handling as the RTP testing groupsexcept that they were tested in isolation. Following these RTP sessions,frontal cortex and parietal cortex gene expression changes in rats weremeasured by microarray. These gene expression changes were examined 6hours after the final of four 30 minutes RTP sessions.

RTP increased expression of the NMDA family of genes (specifically:NMDA. NR1, NR2AD subunits). The upregulation of NMDAR subunits wascorroborated bu qrt-PCR and the results have been presented in the FIG.9.

Evaluation of Hedonic Effect of GLYX-13

To evaluate the hedonic effect of GLYX-13 in this study, the 50-kHzultrasonic vocalisations model of positive affect was examined in rats(see, Burgdorf, J., Panksepp, J., Brudzynski, S. M., Kroes, R. A. &Moskal, J. R. (2008). The effect of selective breeding for differentialrates of 50-kHz ultrasonic vocalizations on emotional behavior in rats.Devel. Psychobiology, 51, 34-46.).

According to 50-kHz ultrasonic vocalizations model of positive affect,RTP behavior in rats has been shown to be rewarding and to elicit highrates of 50kHz ultrasonic vocalizations which in turn have been shown toreflect positive affective states (FIG. 10).

As a part of the study, two groups of three month old FBNF1 rats wereused. The first group was composed of 13 rats and the second group wascomposed of 11 rats. The first group of rats was intravenously injectedwith 10 mg/kg GLYX-13 and the second group of rats was intravenouslyinjected with PBS vehicle (1 ml/kg) in a blind manner via chronicsubdural femoral vein access ports. Both groups of rats were injected 10to 15 minutes before the start of test.

For hedonic ultrasonic vocalizations (USVs), animals received 2 minutesof heterospecific RTP habituation before the final 2 minutes testsession.

Results

Direct injection of GLYX-13 dose (10 mg/kg i.v.) increased hedanic RTPinduced 50-kHz ultrasonic vocalizations. GLYX-13 increased rates ofplay-induced positive affective 50-kHz USVs by 178.5±48.3% (Mean±SEM)compared to vehicle (P<0.01) (FIG. 11).

Discussion

These results suggest that the NMDA receptor may play a functional rolein RTP-induced positive affective states. GLYX-13 may be a usefultherapeutic to increase resilience to depression and anxiety.

Example 9 GLYX-13 Induces Rapid Antidepressant-Like Effects EssentiallyWithout Dissociative Side Effects

Adult male Sprague Dawley rats (Harlan, USA) were used for all studiesexcept for the microinjection studies, for which Fisher Brown Norway F1cross (FBNF1: Harlan, USA) rats were used. All rats were housed inLucite cages with corn cob or sawdust bedding, maintained on a 12:12light:dark cycle (lights on 8 AM), and given ad libitum access to Purinalab chow and tap water throughout the study. All experiments wereapproved by the Northwestern University, New York Medical College, orVirginia Commonwealth University Animal Care and Use Committees.

Porsolt Test

The Porsolt test adapted for use in rats was performed as above. Animalswere placed in a 46 cm tall×20 cm in diameter clear plastic tube filledto 30 cm with tap water (23±1° C.) for 15 min on the first day(habituation) and 5 mm on the subsequent test day.

Microinjection Studies

Bilateral 22-gauge guide cannulae (Plastic Products, USA) werestereotaxically implanted into the infralimbic/prelimbic cortex (+2.7 mmanterior,±1.2 mm lateral, 3.1 mm. ventral to bregma; cannulae angled 12°away from the midline) or dorsal control primary secondary motor cortex.(+2.7 mm anterior,±1.2 mm lateral, 1.0 mm ventral to bregma; cannulaeangled 12° away from the midline). Animals were allowed 1 week torecover from surgery before the start of testing. After the completionof behavioral testing, histology was conducted for cannulae tiplocation. For medial prefrontal cannulae, all tips were located withinthe infralimbic or prelimbic cortex 2.2-3.2 mm anterior to bregma. Formotor cortex cannulae, all tips were located, within the primary orsecondary motor cortex 2.2-3.2 mm anterior to bregma.

Animals were trained with GLYX-13 (10 mg/kg, IV), ketamine (10 mg/kg,IV), or saline vehicle (1 mL/kg, IV) utilizing an unbiased two-chamberconditioned place preference apparatus.

Prepulse Inhibition

Animals were given intravenous injections of GLYX-13 (10 mg/kg) orsaline via chronic indwelling femoral vein cannulae and tested 15 minpost injection, or intraperitoneal ketamine (10 mg/kg) or saline vehicle(1 mL/kg IV or IP respectively) and tested immediately post injectionfor prepulse inhibition.

Drug Discrimination

Testing was conducted as described previously (Nicholson K L & Balster RL (2009) The discriminative stimulus effects of N-methyl-D-aspartateglycine-site ligands in NMDA antagonist-trained rats. Psychopharmacology(Berl) 203(2):441-451). Adult male Sprague Dawley rats were trained todiscriminate ketamine (10 mg/kg IP) from saline. Animals were thentested following various doses of GLYX-13 administration (3-156 mg/kgSC) and the percent of ketamine lever responding and rates of respondingwere recorded. Varions doses of ketamine were tested as a positivecontrol by both the IP and SC routes.

Protein Determinations

Total and Phosphoserine-9 glycogen synthase kinase 3β (GSK-3β) werequantified by ELISA according to the manufacturer's instructions(catalog numbers ADI-900-144 and ADI-900-123A, Assay Designs, USA).Total and phosphoserine-845 GluR1 were quantified by Western blot.

In Vitro Electrophysiology Experiments

Experiments were conducted on hippocampal slices from 14-18 day oldSprague-Dawley rats. Whole cell recordings were obtained from CA1pyramidal neurons voltage clamped at −69 mV, in slices perfused withACSF containing 0 mM [Mg2+] and 3 mM [Ca2+], plus 10 μM bicuculline and20 μM CNQX to pharmacologically isolate NMDAR-dependent. EPSCs.Following bath applications of varying concentrations of GLYX-13 andD-serine, EPSCs were elicited by stimulating Schaffer collateral fiberswith single electrical pulses (80 μs duration) once every 30 s. NMDAREPSCs were characterized by long rise and decay times, and. were fullyblocked at the end of each experiment by bath application of theNMDAR-specific antagonist D-2 -amino-5-phosphonopentanoic acid (D-AP5;50 μM).

Functional Affinity of GLYX-13

Functional affinity, Kp, of GLYX-13 for the NMDAR was estimated usingthe equiactive concentration null method of Stephenson (Stephenson R. P.(1956) A modification of receptor theory. Br J Pharmacol Chemother11(4):379-393) as exemplified by Kenakin (Kenakin T. P. (1997)Pharmacologic Analysis of Drug-Receptor Interaction (New York.)) In thisanalysis concentration-response curves are generated to full agonist,and to the full agonist in the presence of a set concentration of thepartial agonist of interest (FIG. 18, Panel B). Equiactiveconcentrations arc read off the ordinate of each of theconcentration-respouse curves and plotted as the concentration of fullagonist [D-serine] alone and in the presence of partial agonist[GLYX-13] that produce equal effect.

Bioanalysis

Male Sprague Dawley rats (250-300 g) received intravenous injections ofGLYX-13 (3-10 mg/kg), briefly anesthetized with isoflurane, andcerebrospinal fluid (CSF) samples drawn and stored at −70° C. untilanalysis.

CSF samples were precipitated with internal standard Pro-Thr-Pro-Ser-NH2in 400 μl of acetonitrile. Supernatant extract was separated by HPLC(LC-20AD, Shimadzu, Japan) using a Synergi 4 μHydro-RP8A column(Phenomenex, USA) with mobile phase of 100 mM ammonium formate with 0.1%formic acid, for 0.1 minute followed by acetonitrile with 0.1% formicacid and samples were analyzed by LG-MS/MS (API4000, Applied Biosystems,USA) in ESI positive ion mode and the GLYX-13 (199.1 and 216.1) and IS(202.3) daughter ions quantified by multiple reaction monitoring. Thelower limit of quantitation For the assay was 26.3 nM GLYX-13.

Results Behavioral Pharmacology Porsolt Test

GLYX-13 (3-10 mg/kg, IV; 10-30 mg/kg, SC) significantly (P<0.05) reducedfloating times in the Porsolt test by both the IV and SC routs ofadministration as did ketamine (10 mg/kg IP) (FIG. 12A). A scrambledpeptide control (TPTP-NH2; 3 mg/kg IV) had no significant effect (FIG.12B).

FIGS. 12A and 12B show that GLYX-13 produced an acuteantidepressant-like effect in the Porsolt test. Mean (±SEM) time (sec)spent immobile in the Porsolt test in 3-month old male Sprague Dawleyrats were dosed with (A) intravenous GLYX-13 (1, 3, 10, 30 mg/kg),subcutaneous GLYX (1, 3, 10, 30, 56, 100 mg/kg), ketamine (10 mg/kg IP),or saline vehicle; (B) GLYX-13 (3 mg/kg IV), scrambled peptide (3 mg/kgIV) or saline vehicle. All animals were tested 20-60 min post dosing.Animals received a 15-min training swim session one day before dosing.N=7-12 per group. * P<0.05, Fisher's PLSD post hoc vs. vehicle.

MPFC but not motor cortex injections of GLYX-13 (1 and 10 μg/side)reduced floating time in the Porsolt test 20 min post dosing P<0.5)(FIG. 13A). Reduction in floating time was still apparent two weeksfollowing MPFC injection of GLYX-13 (1 μg/side) in an independent groupof animals (P<0.0001) (FIG. 13B).

FIGS. 13A and 13B show that the antidepressant-like effects of GLYX-13were localized to the medial prefrontal cortex. Mean (±SEM) time (sec)spent immobile in the Porsolt test in 3 month old male Sprague Dawleyrats implanted with medial prefrontal or motor cortex (dorsal control)cannulae and injected with GLYX-13 (0.1, 1, 10 μg/side) or sterilesaline vehicle (0.5 μlL/1 min) 20 min (A) or 2 weeks post-dosing (B).Animals received a 15 min training swim session one day before dosing,N=9-10 per group. * P<0.05, Fisher PLSD vs. vehicle.

Drug Discrimination

As shown in FIG. 14, (A) animals treated with SC GLYX-1.3 over the doserange 3-156 mg/kg did not show any ketamine-like discriminative stimuluseffects, whereas animals treated with ketamine IP (5.6-30 mg/kg; P<0.05)or SC (1-10 mg/kg; P<0.05) produced dose-dependent increases inketamine-lever selection (B) GLYX-13 (3-156 mg/kg, SC) did not suppressoperant responding at any dose (all Ps>0.05) unlike ketamine at thehigher doses IP (30 mg/kg; P<0.05) or SC (10 mg/kg; P<0.05).

FIGS. 14A and 14B shows GLYX-13 did not exhibit any ketamine-likediscrimative stimulus effects or suppress operant responding. Mean(±SEM) (A) percentage ketamine-lever responding and (B) rates ofresponding for different doses of ketamine (IP and SC) and GLYX-13 (SC)in rats trained to discriminate 10 mg/kg ketamine, IP, from saline.Values above Sal and Ket are the results of control tests conductedbefore testing each dose response curve. Values above Sal/Sal andSal/Ket are the results of similar control tests performed followingadministration of 2 ml saline SC, 30 min before the session start tomimic conditions of GLYX-13 testing N=6-8 pergroup.

Prepulse Inhibition and Conditioned Place Preference

Ketamine decreased prepulse inhibition (FIG. 15A) an indicator ofdissociative effects, and induced conditioned place preference (FIG.15B) an indicator of abuse potential, all F<0.05. In contrast, GLYX-13did not exhibit either of these effects (FIG. 15) suggesting a lack ofdissociative effects and abuse potential.

FIGS. 15A and 15B shows GLYX-13. did not affect prepulse inhibition, orinduce conditioned place preference. Ketamine (but not GLYX-13)decreased prepulse Inhibition (Panel A), and induced conditioned placepreference (Panel B) in 2-3 month old male Sprague-Dawley rats, N=8-10per group, pretreated with either GLYX-13 (10 mg/kg IV) and tested 15min following injection, or ketamine (10 mg/kg IP) or saline vehicle (1mg/mL IV or IP respectively), tested immediately post injection forprepulse inhibition. N=8-10 per group, * P<0.05, Fisher PLSD vs.vehicle.

Signal Transduction in Response to GLYX-13 or Ketamine

GLYX-13 and ketamine reduced phosposerine-9 GSK-3β/total GSK-3β ratio(P<0.05) (FIG. 14A) and total β-catenin (FIG. 16B). Phosphoserine-845GluR1/total GluR1 ratio was increased in the medial prefrontal cortex,of animals pretreated with. GLYX-13 or ketamine 24 hours prior tosacrifice (<0.05 )(FIG. 16C). As has also been reported for ketamine,the AMPA receptor antagonist NBQX blocked the antidepressant-like effectof GLYX-13 in the Porsolt test (FIG. 16D (P<0.0001).

FIGS. 16A, 16B, 16C, and 16D show GLYX-13 up regulated GluR1Phosphoserine 845 and its antidepressant-like effects are blocked byAMPA receptor antagonism similar to ketamine. Mean (±SEM) (A) Proteinlevels of (A) Phosphoserine 9 GSK-3β/total GSK-3 β, (B) Total Catenin,(C) Phosphoserine 845 GluR1/Total Glur1 in medial prefrontal cortex of 3month old male Sprague Dawley rats dosed with GLYX-13 (3 mg/kg IV),ketamine (10 mg/kg IV) or vehicle 24 hrs before sacrifice; (D) Floatingtime in the Porsolt test in animals pretreated with the AMPA receptorantagonist NBQX (10 mg/kg IP) before GLYX-13 (3 mg/kg IV) dosing andtested 1 hr post dosing, N=4-11 per group, * P<0.05, Fisher PLSD vs.vehicle.

Functional Affinity of GLYX-13 to the NMDA Receptor

Because partial agonists may bind to receptors without producing fulleffects, radioligand binding affinity may reflect artifactually potentaffinity. Thus, the Stephenson method was used to evaluate thefunctional affinity of GLYX-13 for the NMDAR. Precision in thecalculations was improved by repeating the procedure for threeconcentrations of GLYX-13 (FIG. 17, Panel B), from which a series ofpairs of equiactive concentrations of D-serine (ordinate) andD-serine+GLYX-13 (axis) were plotted (FIG. 17, Panel C). Plottinglog(1/slope-1) vs. log [GLYX-13] yielded a line (FIG. 17, Panel D) withpKp as intercept. The use of this method requires that the partialagonist have much less intrinsic activity than full agonists at the sitein order that the ratio εP/εA may be ignored. GLYX-13 was found to have23% of the activity of D-serine in the hippocampal slice assay (FIG. 17,Panels A and B), fulfilling the requirement that partial agonistactivity be 75% or less that of a full agonist, and preferably 25% orless (Kenakin TP (1997) Pharmacologic Analysis of Drug-ReceptorInteraction (New York)).

FIGS. 17A, 17B, 17C, and 17D shows GLYX-13 acted as a partial agonist atthe glycine site of the NMDA receptor. (A-B) Pharmacologically isolatedNMDA receptor current was measured from hippocampal slices to determine(A) the percent intrinsic activity of GLYX-13 as compared to theendogenous ligand which is approximately 23%, and (B) D-Serineconcentration-response in the absence and in the presence of increasingconcentrations of GLYX-13. (C) Plots of equieffective concentrations ofD-Serine in the absence and presence of 1, 10 and 30 μM GLYX-13. (D)Using the slopes (m) from the relationships in Panel b, a plot oflog[1/m)-1] vs. log[GLYX-13] yields x-intercept of log K_(GLYX-13)(Stephenson R P (1956) A modification of receptor theory. Br J PharmacolChemother 11(4):379-393).

Drug distribution studies following IV administration of GLYX-13 at 3and 10 mg/kg revealed C_(max) within the CSF compartment of 0.06 and 0.2μM, respectively (FIG. 18). These concentrations were demonstrated to bepharmacologically relevant in the slice current studies (FIGS. 17A and17B).

FIG. 18 shows mean CSF concentrations GLYX-13 following IVadministration of 3 or 10 mg/kg. N=3 rats/group. GLYX-13 CSF levels weremeasured by LC/MS/MS.

These results show that the NMDA receptor glycine-site partial agonist,GLYX-13, causes a robust, long-lasting antidepressant-like effect withrapid onset in rats. Moreover, at doses that producedantidepressant-like effects, or doses 10-fold higher,GLYX-13, unlikeketamine, essentially did not cause dissociation-like effects in theprepulse inhibition test. GLYX-13 also essentially did not demonstrateketamine-like discriminative stimulus effects or abuse potential in theconditioned place preference test. GLYX-13, 10 mg/kg SC, was active inthe Porsolt test. At the highest dose evaluated, 156 mg/kg SC, in thedrug discrimination assay, GLYX-13 was essentially without effect,making the therapeutic index at least 170:10, or 17.

Equivalents

While specific embodiments of the subject disclosure have beendiscussed, the above specification is illustrative and not restrictive.Many variations of the disclosure will become apparent to those skilledin the art upon review of this specification. The full scope of thedisclosure should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, parameters, descriptive features andso forth used in the specification and claims are to be understood asbeing modified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thisspecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained the presentinvention.

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

What is claimed is:
 1. A method for treating a patient suffering fromdepression, comprising administering an effective amount of a GLXpeptide to said patient.
 2. A method as in claim 1, wherein saiddepression is selected from the group consisting of major depressiveorder, dysthymic disorder, psychotic depression, postpartum depression,seasonal affective disorder, bipoloar disorder, bipolar depression, mooddisorder, depressions caused by chronic medical conditions such ascancer or chronic pain, chemotherapy, chronic stress, post traumaticstress disorders, and manic depressive disorder.
 3. A method as in claim1, wherein the effective amount is about 0.01 mg/kg to about 1000 mg/kgper day.
 4. A method as in claim 1, wherein said GLYX peptide isGLYX-13.
 5. A method for treating anxiety in a patient in need thereof,comprising administering an effective amount of a GLYX peptide to saidpatient.
 6. A method for treating schizophrenia in a patient in needthereof, comprising administering an effecitve amount of a GLYX peptideto said patient.
 7. A method as in claim 5, wherein the effective amountis about 0.01 mg/kg to about 1000 mg/kg per day.
 8. A method as in claim5, wherein the GYLX peptide is GLYX-13.
 9. A method of acutely treatingsymptoms of depression in a patient in need thereof, comprisingadministering an effective amount of GLYX-13 in a single unit dose,whereby said patient is substantially relieved of at least one symptomwithin about 1 day to about 14 days after said administration.
 10. Themethod of claim 9, wherein said patient is substantially relieved of atleast one symptom within about 1 day to about 7 days or less after saidadministration.
 11. The method of claim 9, wherein said patient issubstantially relieved of at least one symptom within about 12 hours toabout 1 day after said administration.
 12. The method of claim 9,wherein said patient is substantially relieved of at least one symptomwithin about 1 hour to about 12 hours after said administration.
 13. Amethod of treating or alleviating depression in a patient in needthereof, comprising administering an effective amount of GLYX-13 to saidpatient, whereby said patient is substantially relieved of at least onesymptom within about 7 days, about 1 day, or about 1 hour after saidadministration.
 14. A method of treating refractory depression in atreatment-resistant patient in need thereof, comprising administratingan effective dose of GLYX-13 to said patient, whereby said patient issubstantially relieved of at least one symptom within about 14 days. 15.The method of claim 14, wherein the treatment-resistant patient isidentified as one who has been treated with at least two types ofantidepressant treatments prior to administration of GLYC-13.
 16. Themethod of claim 14, wherein the treatment-resistant patient isidentified as one who is unwilling or unable to tolerate a side effectof at least one type of antidepressant treatment.
 17. The method ofclaim 1, wherein the GLYX peptide is represented by:

pharmaceutically acceptable salts, homologs, or hydrates thereof. 18.The method of claim 17, wherein the GLYX peptide is the acetate salt.